Interpretive Summary: Allergy to peanuts and tree nuts appears to be increasing in frequency, and strict avoidance is the only accepted treatment
currently. Cashew nut allergens are often a cause of very severe reactions, and cashew allergens survive several types of
processing. There are three cashew allergens characterized in the literature; and we examined the ability of stomach and
intestinal proteases to digest these allergens using a model system. One of the cashew allergens in particular, Ana o 3, was able
to resist digestion by both stomach and intestinal proteases better than the others. We found conditions that increased the
ability of Ana o 3 to be digested, and these findings may lead to better processing methods that can reduce or eliminate the ability
of cashew allergens to cause severe allergic reactions.

Technical Abstract:
The stability of food allergens to digestion varies; and the ability of food proteins to cause an allergic reaction may be affected by the susceptibility of the allergen to digestion by proteases, including pepsin and trypsin. Recent studies have demonstrated that cashew nut allergens are often a cause of anaphylaxis, and cashew allergens are stable to several types of processing. Three conserved cashew allergens have been described; including the 7S globulin Ana o 1, 11S globulin Ana o 2, and the 2S albumin Ana o 3. We characterized the stability of cashew allergens to digestion by pepsin and trypsin in-vitro and identified IgE binding epitopes that survive digestion. Samples were evaluated by SDS-PAGE, mass-spectrometry, and immunoblotting to compare IgE
binding. Increasing protease activity resulted in greater degradation of higher molecular weight cashew proteins. Among cashew
proteins, the 2S albumin, Ana o 3, was most resistant to digestion by both pepsin and trypsin. Mass-spectrometry identified
digestion resistant Ana o 3 protein fragments that retained reported IgE binding epitopes. Pre-treatment of extracts or purified
Ana o 3 with reducing agent increased the sensitivity of Ana o 3 to protease digestion. Circular dichroism revealed the structure
of purified Ana o 3 was largely alpha-helical and was disrupted following reduction. Ana o 3 reduction followed by digestion with
trypsin decreased binding of serum IgE from cashew allergic patients. Our results indicate that the Ana o 3 disulfide bond-dependent structure protects the protein from proteolysis, and that Ana o 3 is the cashew allergen most likely to survive
gastrointestinal digestion intact.